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HF MicroBrx™
User Guide
Introduction
2
HF MicroBrx Design
3
One Bioreactor … Two Compartments
ExtraCapillary (EC) Space
3
IntraCapillary (IC) Space
3
Culture Respiration
4
Hollow Fiber Membranes are Porous
4
Nutrient Delivery and Metabolic Waste Removal
4
HF MicroBrx Culture Maintenance
Temperature and pH Control
5
HF MicroBrx Usage
Time Requirements
Material Requirements
5
5
Day 1 Activities
Check Kit Contents
Preparation
Flushing HF MicroBrx
Flush the EC
Flush the IC
Drain and Refill the EC
Experimental Goals
Cell Preparation
Inoculation
6
6
7
7
8
9
10
10
10
Days 2, 3, and 4 Activities
Culture Maintenance and Testing
Harvest and Count Cells
11
11
Discussion
Cell-Side Medium
Non Cell-Side Medium
Cell Type
Interpretation of Low Density
Inoculation Results
Interpretation of High Density
Inoculation Results
12
12
12
12
12
Let Us Know How It Went!
13
Publications with HF MicroBrx™
13
Ordering Information
13
Introduction
Never tried hollow fiber technology before?
Pursuing R&D or feasibility projects?
Don’t use full-scale hollow fiber bioreactors that are
bigger than necessary and support very large culture
volumes.
Use HF MicroBrx™, our micro-sized hollow fiber
bioreactor for your smallest-scale needs. HF MicroBrx
does not need custom equipment either. It simply goes
in your own CO2 incubator.
This kit contains the tools you need to experience the
benefits of hollow fiber cell culture. Please send us your
feedback about using HF MicroBrx!
HF MicroBrx was designed by Biovest International as a
screening tool for hollow fiber bioreactor R&D. We
published several papers featuring HF MicroBrx in peerreviewed journals1-5. These papers illustrate the
usefulness of HF MicroBrx for screening medium
components and subclones to optimize the
performance of your hollow fiber bioreactor system.
HF MicroBrx is being commercialized and is now
available to help you experience the advantages of
ultra-high-density cell growth.
Biovest’s HF MicroBrx™: 0.2 mL culture volume, 30 fibers
Biovest’s full-scale hollow fiber bioreactor: 155 mL culture volume, 1000’s of fibers
2
HF MicroBrx™ User Guide
© Biovest International, Inc
700537-000 Rev. A
HF MicroBrx Design
One Bioreactor … Two Compartments
HF MicroBrx, like our full-scale hollow fiber bioreactor
systems, has two compartments that are separated from
one another by the semi-permeable membrane (wall) of
the hollow fiber
ExtraCapillary (EC) Space
•
•
•
•
•
•
Volume outside of the hollow fibers
Large volume, ~4.5 mL
Functions as reservoir of small nutrients
Cell culture-free (non cell-side of hollow fiber wall)
Product-free
Cell culture medium in EC may or may not
contain growth supplements
EC Port
(non cell-side)
IC Port
(cell-side)
Longitudinal and cross sections showing one of
two identical ends of HF MicroBrx
IntraCapillary (IC) Space
•
•
•
•
•
Combined volume inside all of the hollow fibers
Small volume, ~0.2 mL
Contains cell culture (cell-side of hollow fiber wall)
Contains product
Cell culture medium in IC contains growth
supplements (unless protein-free adapted) .
EC
IC
Hollow Fiber
Wall
IC is open
through the glue
EC stops…
where the
glue surrounds
the fibers
Cross Section of a hollow fiber.
Cells and product remain in the IC.
Basal medium in the EC is the nutrient reservoir.
EC Medium omitted at right for clarity.
HF MicroBrx™ User Guide © Biovest International, Inc
700537-000 Rev. A
3
Culture Respiration
O2 and CO2 move through the materials of
HF MicroBrx and the cell culture medium
to facilitate cellular respiration:
O2 and CO2 freely:
1) diffuse through silicone tubing
2) dissolve into cell culture medium
3) diffuse through the hollow fiber wall
(membrane)
O2 moves toward the cell culture from the
incubator air.
CO2 enters the EC medium from the incubator
gases or from the cell culture inside the fibers.
Silicone Tubing
Hollow Fiber Bundle
O2
Diffusion
Hollow Fiber Membranes are Porous
CO2
Exchange
• ~60 KDa MWCO
• Small nutrients (O2, glucose, etc.) and
metabolic wastes (CO2, lactate, etc.) easily
pass through
• Cells stay in the IC space
• Most secreted proteins stay and concentrate
in the IC space
• Most protein supplements stay in the IC
space, resulting in very little usage of these
expensive components!
Nutrient Delivery and Metabolic Waste
Removal
The EC space is an ~4.5 mL reservoir of
basal medium nutrients that are small
molecules that readily pass through
the pores in the hollow fiber wall (filter
membrane). These nutrients diffuse
toward the cell culture and are
metabolized.
Metabolic wastes, such as lactate and
ammonia, also are small molecules
that readily pass through the pores in
the hollow fiber wall, diffuse away
from the cell culture, and accumulate
in the basal medium in the EC space.
4
HF MicroBrx™ User Guide
Glucose
O2
Lactate
CO2
© Biovest International, Inc
700537-000 Rev. A
HF MicroBrx Culture Maintenance
Temperature and pH Control
Keep HF MicroBrx into a standard CO2
incubator to maintain temperature and pH.
pH control is generally accomplished over the
course of the experiment using cell culture
medium that contains at least 2 g/L sodium
bicarbonate, NaHCO3. If pH drops too much,
however, increase NaHCO3 concentration to
3.5 g/L or higher.
Incubator
37°C, 5% CO2
HEPES buffer is not recommended as it can
interfere with the NaHCO3 buffering system.
To monitor pH during the experiment, remove a
very small volume of EC medium and measure
pH offline. There are practical limitations to
measuring pH offline because of the small total
volumes HF MicroBrx contains and how quickly
pH in the medium sample can change.
Contact Biovest’s Account Services group, if you want to measure pH offline or adjust pH during the
course of the experiment by regulating the incubator’s %CO2 setpoint .
HF MicroBrx Usage
Follow these procedures to use HF MicroBrx. For more information, contact Biovest’s Account Services
group and see the referenced publications.
Use only sterile supplies and perform all tasks aseptically in a sterile hood!
Time Requirements
Material Requirements
Total time required to complete this
experiment is approximately:
2 hours of cell culture
60 – 100 mL cell culture medium per HF MicroBrx
2 hours of bioreactor work
2 hours of analysis
0.25 – 1 x 108 cells per HF MicroBrx
Optional: pH meter, glucose and/or lactate assay
Miscellaneous cell culture supplies
You may need to purchase additional syringes
depending on your technique and application.
HF MicroBrx™ User Guide © Biovest International, Inc
700537-000 Rev. A
5
Day 1 Activities
Check Kit Contents
Standard Kit
1 Bag (as shown at right)
15 3-mL Syringes (not shown)
12 20-mL Syringes (not shown)
Sterile bag of HF MicroBrxs contains:
3
12
High-Volume Kit
4 Bags (as shown at right)
6
0 3-mL Syringes (user-provided)
0 20-mL Syringes (user-provided) 6
Preparation
Remove Vent Plugs,
Save,
Keep Sterile
1. Open outer, non-sterile bag.
2. Remove and set aside all syringes.
Tight !
3. Remove sterile, sealed bag that contains
MicroBrxs, dilators, red and green caps.
4. Examine integrity of sterile, sealed bag. If
damaged, contact Biovest.
Remove, Save,
Keep Sterile,
Put on Vent Plugs
5. Disinfect outside of sterile, sealed bag with
70% isopropanol, or equivalent.
6. Place sterile, sealed bag in a sterile laminar
hood.
7. Open sterile, sealed bag.
Use these tubing lines
later on with aseptic
handling. They can be
autoclaved if necessary.
8. Remove MicroBrxs, dilators, red and green
male luer plugs.
9. Confirm all Kit Contents are present.
10. Replace natural-colored, vented caps with
colored caps:
• green caps go on both side ports.
• red caps go on both end ports.
• put vent plugs in short tubing
• repeat for all MicroBrxs.
6
HF MicroBrx™ User Guide
11. Ensure natural-colored double female luer
fitting connected to red luer of IC port is tight
so it will not come off when the naturalcolored cap is removed in the next step.
12. Ensure each HF MicroBrx looks like the
illustration below before continuing..
© Biovest International, Inc
700537-000 Rev. A
Day 1 Activities
Flushing HF MicroBrx
Flush the outside (EC) and inside (IC) of the hollow fibers to remove their wetting agent, which is
cytotoxic. Do not skip these steps!
Flush the EC
1. Prepare 140 mL of growth-supplemented
medium and warm it to 37°C.
5. Disconnect the other green cap from the other
EC Port and keep cap sterile!
2. Fill 20-mL syringe with medium.
2a. Use the 10 cm-long dilator to simplify
aseptically filling syringe.
6. Connect empty 20-mL syringe to open EC Port.
7. Hold HF MicroBrx at ~45° angle with filled
syringe at lower end.
8. Inject 20 mL through EC. Simultaneously:
8a. ~15.5 mL flows into empty 20-mL syringe
8b. ~4.5 mL remains in EC of HF MicroBrx
Do not leave air bubbles in EC
2b. Remove the sterile cap and sterile tubing
cover before using the dilator.
9. Replace syringes with sterile, green caps.
10. Repeat steps 1–9 for the other two
HF MicroBrxs.
3. Disconnect one green cap from either one of
the side ports (EC Ports) and keep cap sterile!
4. Connect filled syringe to open EC port.
Sterile Hood
Suggestion:
Use sterile tubing
to temporarily
cover green caps
9b
2
20 mL
.5
~4
9b
3
4
45°
1
140 mL, 37°C
6
9a
Flush the EC
ai
m
e
Lr
m
5
ns
8
~15.5 mL
In
ll
Pu
mL
Push Out 20
8
9a
7
HF MicroBrx™ User Guide © Biovest International, Inc
700537-000 Rev. A
7
Day 1 Activities
Flush the IC
3. Connect filled syringe to open IC port.
6. Caution Notes!
a) Step 7 requires ~15 seconds. Do not inject
too quickly or the fibers may burst!
b) During step 7 observe the medium as it
flows through the inside of the hollow fibers.
Inject enough medium that the air bubbles
inside the fibers are eliminated.
4. Disconnect the other red cap from the other IC
Port and keep cap sterile!
7. Inject medium through IC and collect ≥1 mL in
empty syringe.
5. Connect empty 3-mL syringe to open IC Port.
8. Replace syringes with sterile, red caps.
1. Fill 3-mL syringe with 37°C
growth-supplemented medium using dilator
as previously shown (not illustrated below).
2. Disconnect one red cap from either one of the
end ports (IC Ports) and keep cap sterile!
9. Repeat steps 1–9 for the other two
HF MicroBrxs.
10. Place MicroBrxs in a 37°C, 10%*, humidified
CO2 cell culture incubator for ≥4 hours.
(* some media may require a higher or lower
%CO2 to achieve the desired pH.)
Sterile Hood
Suggestion:
Use sterile tubing
to temporarily
cover red caps
9
7
2 mL
Push Out ~
conds
over 15 se
5
1
2
3 mL
6
emains
~0.2 mL r
Bubbles
Clear All
ers!
Inside Fib
3
4
8
Flush the IC
8
HF MicroBrx™ User Guide
© Biovest International, Inc
700537-000 Rev. A
.8 mL
Pull In ~1 nds
eco
over 15 s
5
Day 1 Activities
Drain and Refill the EC
1. Disinfect the outside of the HF MicroBrxs and
place them in a sterile laminar hood.
8. Connect syringe with 20 mL fresh medium to
open EC Port.
2. Fill 20-mL syringe with 37°C growthsupplemented medium (for step 8).
9. Hold HF MicroBrx at ~45° angle with full
syringe at lower end.
3. Disconnect one green cap from either EC Port
and keep cap sterile!
10. Inject 20 mL into EC: ~15.5 mL flows into
partially filled syringe. ~4.5 mL remains in EC of
HF MicroBrx. Do not leave air bubbles in EC.
4. Connect an empty 20-mL syringe to open EC
port.
11. Replace syringes with sterile, green caps.
5. Disconnect the other green cap from the other
EC Port and keep cap sterile!
12. Repeat steps 1–11 for the other two
HF MicroBrxs.
6. Hold HF MicroBrx at ~45° angle with empty
syringe at lower end.
13. HF MicroBrxs are ready for inoculation.
7. Pull ~4.5 mL flush medium from EC into empty
syringe.
Suggestion:
Use sterile tubing
to temporarily
cover green caps
Sterile Hood
6
Step 1: Drain the EC
as shown here
3
Step 2: Flush the EC
as previously shown
8
1
45°
7
2
L
Pull Out ~4.5 m
5
4
HF MicroBrx™ User Guide © Biovest International, Inc
700537-000 Rev. A
9
Day 1 Activities
Experimental Goals
Subsequent instructions describe evaluating the results from low-density and high-density inoculation
conditions. Low-density inoculation provides insight to the growth phase of a full-scale bioreactor run.
High-density inoculation provides insight to the production phase of a full-scale bioreactor run. If your
experimental goals differ, use these instructions as a guideline and adjust them accordingly.
Cell Preparation
1. Harvest cells already expanded in T-flasks or spinners: need ~200 mL at 5x105/mL (1x108 cells).
2. Centrifuge cells.
3. High-Density Inoculum, 5x107/mL:
Resuspend cells in ~2 ml of fresh, 37°C medium.
4. Low-Density Inoculum, 5x106/mL:
Make a 1:10 dilution of high-density inoculum (0.5 ml cells, 4.5 ml fresh medium).
5. Save a small sample of each inoculum to determine actual inoculation densities.
Inoculation
1. Disinfect IC Ports of HF MicroBrxs and place in sterile laminar hood.
2. Attach 10-cm dilator to 3-mL syringe.
3. Fill syringe with Low- or High-Density Inoculum.
4. Discard dilator.
5. Disconnect one red cap from an end port (an IC Port), carefully set it aside, and keep cap sterile!
6. Connect syringe to open IC port.
7. Disconnect other red cap, carefully set it aside, and keep cap sterile!
8. Connect empty 3-mL syringe to open IC port.
9. Inject 0.5 – 1 ml of cells through IC. Slowly push on full syringe while slowly pulling at the same rate
on empty syringe.
10. Remove syringes and keep them sterile.
11. Reconnect red caps.
12. Repeat steps 5 – 10 with remaining HF MicroBrxs using remaining Low- or High-Density Inoculum.
10
HF MicroBrx™ User Guide
© Biovest International, Inc
700537-000 Rev. A
Days 2, 3, and 4 Activities
Culture Maintenance and Testing
1. Place HF MicroBrxs in CO2 incubator for 3 days. Longer culture times may be necessary depending on
your cell line or application.
2. Follow culture’s growth by daily analysis of glucose in a small sample of EC medium, if you have the
ability to assay this metabolite using only very a small sample volume.
3. Drain and refill EC medium if glucose concentration is 50 – 60% of starting value:
a) Low-density inoculation should not require EC medium exchanges.
b) High-density inoculation may require daily EC medium exchanges.
Harvest and Count Cells
Perform these steps aseptically if the experiment will continue—to, for example, reclone the cells to
select for a higher-secreting cell line that is better acclimated to high-density culture conditions. If only
cell counts and assays will be performed, aseptic handling may be unnecessary.
1. Fill sterile 3-mL syringe with air.
7. Since the IC is only 0.2 ml, very little volume is
harvested — usually 0.05 to 0.15 mL.
2. Remove red cap from either IC port.
3. Connect air-filled syringe to open IC port.
4. Disconnect double female luer fitting from IC
port on opposite end.
5. Hold open IC end over microcentrifuge tube.
6. In one quick motion, inject air into IC while
collecting effluent in microcentrifuge tube.
8. The IC harvest is generally enough to get good
cell counts and determine product titer. For
attachment dependent cells, it may be helpful
to determine if any cells still remain in the
bioreactor by passing a trypsin solution
through the IC space.
Sterile Hood
3
4
1
Push in air
4
2
3 mL air
Disconnect both
pieces before
harvesting!
<200 µL
Harvest Cells
and Product
HF MicroBrx™ User Guide © Biovest International, Inc
700537-000 Rev. A
11
Discussion
Cell-Side Medium
Cell-side medium used in hollow fiber systems is typically the same medium used in T-flask cultures —
basal medium with added growth supplements or a defined serum-free medium.
In some cases, cell-side medium requires additional supplementation of specific components that are too
large to cross the fiber from cell-side to non cell-side.
Until you have experience using hollow fiber technology, we suggest that you simply use routine T-flask
medium for your HF MicroBrx study.
Non Cell-Side Medium
In many cases, non cell-side medium can be simply basal medium (no growth-supplements).
However, in some cases, cell growth is slow using just basal medium on the non cell-side. For this reason,
we recommend that you use the same (complete) medium on the non cell-side and the cell-side that you
use for routine T-flask cultures.
Cell Type
A wide range of cell types have been successfully cultured in hollow fiber bioreactors. Suspension
adapted cells typically grow faster initially compared to attachment-dependent cells.
Interpretation of Low Density Inoculation Results
The low-density inoculation results have been very useful for predicting initial growth of a cell line in a
large scale hollow fiber system. If the cell line grows well in HF MicroBrx, then you should expect good
growth of that cell line in our large scale systems. Good growth would be an expansion from ~ 5 million
per mL to 10-50 million per mL over the 3-day study.
Interpretation of High Density Inoculation Results
The high-density results are intended to mimic the production phase of hollow fiber bioreactors.
However, our results demonstrate that cell growth is limited by both oxygen and medium in HF MicroBrx,
limiting the viable cell density to about 5-10 x 107/mL (whereas the viable cell density in a large-scale
hollow fiber bioreactor approaches the mid 108/mL range). Despite these limitations, it is instructive to
experience the high cell density and high product titers associated with high density inoculation.
12
HF MicroBrx™ User Guide
© Biovest International, Inc
700537-000 Rev. A
Let Us Know How It Went!
We are interested in what you think about HF MicroBrx and are willing to discuss the significance of your
results. We have solved poor growth problems for a number of cell lines using HF MicroBrx. Some of the
variables we have looked at include cell-side medium formulation, non cell-side medium formulation,
and subclone selection. For discussion of your results, please contact:
Curt Gleiter
Director of Technical Services
[email protected]
Phone: 763-786-0302
Biovest International, Inc.
8500 Evergreen Blvd.
Minneapolis, MN 55433
biovest.com
Publications with HF MicroBrx™
Gramer, M. J., Maas, J. (2003) Optimal NS0 cell growth in a hollow fiber bioreactor requires increased
serum concentration or a cholesterol supplement on the cell side of the fiber. Biotechnology Prog.,
19, 1762 - 1766.
Gramer, M.J., Britton, T.B. (2002) Antibody production by a hybridoma cell line at high cell density is
limited by two independent mechanisms. Biotech. Bioeng., 79, 277 - 283.
Gramer, M.J., Britton, T.B. (2000) Selection and isolation of cells for optimal growth in hollow fiber
bioreactors. Hybridoma., 19, 407 - 412.
Gramer, M.J., Poeschl, D.P. (2000) Comparison of cell growth in T-flasks, in micro hollow fiber bioreactors,
and in an industrial hollow fiber bioreactor reactor system. Cytotechnology., 34, 111 - 119.
Gramer, M.J., Poeschl, D.P. (1998) Screening tool for hollow fiber bioreactor process development.
Biotech. Prog., 14, 203 - 209.
Ordering Information
Order part number 600322-000 for the Standard HF MicroBrx Kit
Order part number 600322-001 for the High-Volume HF MicroBrx Kit
8500 Evergreen Boulevard
Minneapolis, MN 55433-6000 USA
www.biovest.com
Telephone: (763) 786-0302
Toll-free in the U.S.: (800) 325-1112
After Hours: (800) 325-1112 (voice prompt)
Fax: (763) 786-0915
[email protected]